Substance P (SP) is an 11 amino acid peptide that plays multiple physiological and pathological roles as a neurotransmitter, an autocrine agent, and a neurohormone. SP participates in diverse functions such as the transmission of pain, regulation of inflammation, and in mediating behavioral responses. The activities of SP depend upon interaction with its 7-transmembrane G-protein coupled receptor, the NK1 receptor (NK1R). The NH2 terminus has two consensus sequences for N glycosylation and there is good evidence that the NH2 terminus is glycosylated; however little is known of the chemical structures of the attached carbohydrates nor their function. This proposal addresses this gap in our knowledge and focuses on the chemistry and biological consequences of NK1R glycosylation. HT29 cells (human colonic cells) will be stably transfected with cDNA encoding wild type human NK1 receptors or mutant receptors with one, the other or both N-glycosylation sites altered. Methods for purification of receptors based on his-tagged techniques will be developed to extract these receptors in sufficient quantity and purity to permit mass spectrometric analysis of their carbohydrate moities. Several functional studies will be done to assess possible differences in wild type and mutant receptors including comparing a) association and disassociation rates of ligand binding to receptors; b) receptor internalization; c) and signal transduction mechanisms including activation of cAMP, phosphoinositide hydrolysis and transactivation of the MARK pathway. These pilot experiments may lead to new directions for understanding the role of glycosylation in SP-NK1R mediated responses that could be of therapeutic importance.